Skip to main content
SLU publication database (SLUpub)

Research article2002Peer reviewed

Rat mast cell protease 4 is a beta-chymase with unusually stringent substrate recognition profile

Karlson, Ulrika; Pejler, Gunnar; Fröman, Gunnar; Hellman, Lars

Abstract

Activated mast cells release a variety of potent inflammatory mediators including histamine, cytokines, proteoglycans, and serine proteases. The serine proteases belong to either the chymase (chymotrypsin-like substrate specificity) or tryptase (trypsin-like specificity) family. In this report we have investigated the substrate specificity of a recently identified mast cell protease, rat mast cell protease-4 (rMCP-4). Based on structural homology, rMCP-4 is predicted to belong to the chymase family, although rMCP-4 has previously not been characterized at the protein level. rMCP-4 was expressed with an N-terminal His tag followed by an enterokinase site substituting for the native activation peptide. The enterokinase-cleaved fusion protein was labeled by diisopropyl fluorophosphate, demonstrating that it is an active serine protease. Moreover, rMCP-4 hydrolyzed MeO-Suc-Arg-Ala-Tyr-pNA, thus verifying that this protease belongs to the chymase family. rMCP-4 bound to heparin, and the enzymatic activity toward MeO-Suc-Arg-Ala-Tyr-pNA was strongly enhanced in the presence of heparin. Detailed analysis of the substrate specificity was performed using peptide phage display technique. After six rounds of amplification a consensus sequence, Leu-Val-Trp-Phe-Arg-Gly, was obtained. The corresponding peptide was synthesized, and rMCP-4 was shown to cleave only the Phe-Arg bond in this peptide. This demonstrates that rMCP-4 displays a striking preference for bulky/aromatic amino acid residues in both the P1 and P2 positions.

Published in

Journal of Biological Chemistry
2002, volume: 277, number: 21, pages: 18579-18585
Publisher: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

SLU Authors

  • Pejler, Gunnar

    • Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences

Associated SLU-program

Future Animal Health and Welfare (until Jan 2017)

UKÄ Subject classification

Biochemistry and Molecular Biology

Publication identifier

  • DOI: https://doi.org/10.1074/jbc.M110356200

Permanent link to this page (URI)

https://res.slu.se/id/publ/66602